Abstract Defective intestinal epithelial tight junction (TJ) barrier has been postulated to be a key pathogenic factor in variety of inflammatory diseases of the gut (including Crohn's disease, ulcerative colitis, necrotizing enterocolitis, NSAID associated enteritis, and infectious diarrheal syndromes) and systemic inflammatory conditions (including alcoholic liver disease, heat stroke, hemorrhagic shock, and critically ill). The defective intestinal TJ barrier allows paracellular permeation of luminal antigens across the epithelial barrier, leading to both local inflammatory response in the gut and systemic circulation of bacterial endotoxins culminating in systemic inflammatory response and end-organ damage. Although gut-derived lipopolysaccharides play a critical role in the pathogenesis of intestinal inflammation and systemic inflammatory response, their role in disruption of intestinal epithelial tight junction barrier remains unknown. Our preliminary studies indicated that lipopolysaccharide (LPS) at physiologically relevant concentrations (.1 to 1 ng/ml) causes an increase in intestinal epithelial TJ permeability, unrelated to apoptosis or cell death. The broad objectives of this grant proposal are to elucidate the intracellular and molecular mechanisms that mediate the LPS induced increase in intestinal TJ permeability using an-in-vitro (consisting of filter-grown Caco-2 intestinal epithelial monolayers) and in-vivo (re-cycling intestinal perfusion) murine model systems. Our preliminary data suggested that the LPS induced increase in intestinal epithelial TJ permeability was mediated in part by: 1) increase in Toll-like receptor-4 (TLR-4) expression; 2) activation of p38 kinase pathway; and 3) increase in myosin light chain kinase (MLCK) mRNA and protein expression. Based on our preliminary studies, we hypothesize that physiologically relevant concentrations of LPS induce an increase in intestinal epithelial TJ permeability via TLR-4 signal transduction cascade activation of MLCK gene and MLCK-regulated opening of the intestinal epithelial TJ barrier. The proposed specific aims are to: 1) delineate the involvement of TLR-4 signal transduction cascade in LPS induced increase in intestinal epithelial TJ permeability; 2) delineate the molecular processes involved in LPS induced increase in intestinal TJ permeability; and 3) delineate the mechanisms involved in LPS induced increase in intestinal permeability in-vivo.